Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP

Abstract

Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1μg/mL and removing doxycycline from the media 60min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.